The present invention relates to a process for analyzing a mass spectrum obtained by a mass spectrometer, and more particularly to a method and apparatus for measuring and analyzing a mass spectrum which are optimum to determine a molecular ion and hence the molecular weight from a mass spectrum resulted under atmospheric pressure ionization.
Mass spectrometry is not only a measuring process with very high sensitivity, but also a superior analyzing process capable of providing the molecular weight and structural information of a sample. Also, an apparatus in which separating means, e.g., a gas chromatograph or liquid chromatograph, is provided as a preceding stage of a mass spectrometer to directly separate and analyze a mixture has been developed and widely used in recent years.
In general, a sample molecule introduced to a mass spectrometer is first ionized by an ion source, dispersed in a mass spectrometry section depending on its mass, and detected by a detector, thus providing a mass spectrum.
On that occasion, a resulting mass spectrum is usually represented in the form of a bar graph. In a bar graph, for example, a horizontal axis (X-axis) indicates a mass to charge ratio (m/z) of each ion and a vertical axis (Y-axis) indicates relative intensity of each ion that is normalized with the strongest mass peak as 100%.
A mass spectrum is made up of a molecular ion (having an m/z value of, for example, 200) resulted from ionization of a molecule itself, fragment ions (plural ions each having an m/z value of, for example, not larger than 200) resulted from a molecular ion being fragmented, an adduct ion (having an m/z value of, for example, larger than 200), and so on.
At the present, the Atmospheric Pressure Chemical Ionization (APCI) method, the Electro-Spray Ionization (ESI) method, etc. are employed in many cases. In both of those methods, because of soft ionization (meaning ionization that occurs with small energy), fragment ions are hard to be produced, while a quasi-molecular ion resulted from a proton or an alkali-metal ion (e.g., an Na.sup.+ ion) being attached to a molecule is produced with high intensity. By using those methods, therefore, an unstable compound or the like can also be stably ionized.
In those atmospheric pressure ionization methods, because ionization takes place under the atmospheric pressure, the produced ion still exists under the atmospheric pressure after the ionization. The produced ion therefore repeatedly collides with neutral molecules (solvent molecules in many cases) in the surroundings. An ion produced under the atmospheric pressure is introduced to a chamber under vacuum for mass spectrometry. Upon being thus introduced to the vacuum chamber, the produced ion is quickly cooled owing to abrupt expansion (adiabatic expansion).
Once a solvent molecule which has been cooled similarly to a cooled ion collides with the ion, the solvent molecule can no longer detach from the ion and therefore an adduct ion including at lease one of many polar molecules (solvent molecules) attached thereto is created. The creation of such an adduct ion lowers the intensity of a molecule ion because an ion which should form a single mass peak (molecular ion) in its ideal condition is dispersed to a plurality of adduct ions.
As a result, the SN ratio of a molecule ion and hence the detection sensitivity of an apparatus are apparently reduced to a large extent. Also, a mass spectrum becomes complicated and the analyzing process is impeded by the emergence of an adduct ion. For this reason, commercially available LC/MS systems include means for dissociating adduct ions (by heating, activation upon collision of ions, and so on). Hence, the intensity of adduct ions is generally very small in mass spectra obtained by commercially available LC/MS systems.
Checking mass spectra in detail, however, those adduct ions sill have intensity at an observable level in many cases.
The attachment energy with which a polar molecule is attached to an ion is usually 1 eV or below that is much smaller than the bond energy of a chemical bond (such as a C --C or C--H bond) constituting an ion.
Accordingly, when a molecular ion is subject to excessive energy (heating, acceleration or collision of the ion, etc.) enough to produce fragment ions, a molecule attached to the molecular ion is first dissociated. After that, a covalent bond is disconnected to produce fragment ions. Therefore, an adduct ion made up of a fragment ion and a polar ion attached to it does not exist usually.
Because of soft ionization, the atmospheric pressure ionization gives a simple mass spectrum in which fragment ions are few and a quasi-molecular ion (protonated ion) is emphasized. Conversely, simplicity of a mass spectrum is often not enough to provide a conclusive factor in determining the molecular weight.
In the Electron Ionization (EI) method which produces many fragment ions, mass differences between an ion estimated to be a molecular ion and a plurality of fragment ions are determined, and the molecular ion is estimated without including contradictions in comparison with the process where fragments are created (called fragmentation). As more fragment ions can be explained without including contradictions, the more exact is the estimation.
However, mass spectra obtained by LC/MS systems are generally very simple in many cases. In such a mass spectrum, it is difficult to immediately judge whether the produced ion is a quasi-molecular ion, or a fragment ion resulted from the quasi-molecular ion being fragmented, or an adduct ion having a solvent molecule attached thereto. To estimate the quasi-molecular ion, therefore, its molecular weight must be estimated through a complicated try-and-error process below.
Specifically, first, the strongest mass peak in a high molecular region of a resulting mass spectrum is assumed to represent a quasi-molecular ion. Then, mass differences between the assumed quasi-molecular ion corresponding to the strongest mass peak and ions around the strongest mass peak are determined. After that, the mass differences are checked one by one on whether it agrees with the molecular weight of any of solvent molecules and an ammonium ion. If agreement is found, it is judged that the assumed quasi-molecular ion is possibly an adduct ion.
Next, by assuming another mass peak to represent a quasi-molecular ion, the quasi-molecular ion is estimated through a similar process as mentioned above. As a result of such repeated try-and-error processes, one of the assumed quasi-molecular ions which accompanies minimum contradictions is estimated to be a quasi-molecular ion of target.
However, the above estimate analysis is made by a man such as a person in charge of analysis and a process of the estimate analysis is merely a repetition of try-and-error operations. This means that an incorrect prediction tends to easily mix in the estimation or assumption, and that a missing or misunderstanding is more likely to occur as a matter of course. Eventually, a possibility of mistakes or overlooks becomes very high. Also, a lot of time and labor are required.
A possibility as to whether an adduct ion or the like emerges or not greatly depends on physicochemical properties of a compound, LC analyzing conditions (such as the kind, pH, flow rate and temperature of an eluent), measuring conditions of an LC/MS system (such as the ionization method, i.e., ESI or APCI, and the voltage, temperature and pressure of an interface), etc. The analysis of a mass spectrum must be made in consideration of those preconditions as well.
Accordingly, the analysis of a mass spectrum of unknown components requires a high level of knowledge and long experiences. Since a person in charge of measurement and a person in charge of analysis are generally different from each other in many cases, it is unavoidable that all the analyzing conditions are not exactly informed from the person in charge of measurement to the person in charge of analysis, and therefore an error is brought into the process of analysis.
Further, when a plurality of components are eluted from an LC in overlapped relation, or when a sample to be measured is a mixture, a mass spectrum is given by a combination of spectra corresponding to those plural components or several ingredients of the mixture. In such a case, a resulting mass spectrum is so complicated that it is difficult for even an experienced person to analyze the mass spectrum.
In the measurement using an LC/MS system, the system is usually operated to provide mass spectra not less than 1000 per day. Analysis of mass spectra in such a large number requires a lot of time and labor; hence a longer time than necessary for the measurement must be taken for the analysis. Accordingly, an error is more likely to occur in the process of analysis as a matter of course. This has been a big obstacle in improving the efficiency of qualitative analysis using LC/MS systems.